Mitochondrial Glutamate Dehydrogenase Research Articles

Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity.

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids towards ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH knockout mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e. suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH knockout mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

Cryo-EM structure of the KLHL22 E3 ligase bound to an oligomeric metabolic enzyme

CULLIN-RING ligases constitute the largest group of E3 ubiquitin ligases. While some CULLIN family members recruit adapters before engaging further with different substrate receptors, homo-dimeric BTB-Kelch family proteins combine adapter and substrate receptor into a single polypeptide for the CULLIN3 family. However, the entire structural assembly and molecular details have not been elucidated to date. Here, we present a cryo-EM structure of the CULLIN3RBX1 in complex with Kelch-like protein 22 (KLHL22) and a mitochondrial glutamate dehydrogenase complex I (GDH1) at 3.06Å resolution. The structure adopts a W-shaped architecture formed by E3 ligase dimers. Three CULLIN3KLHL22-RBX1 dimers were found to bedynamically associated with a single GDH1 hexamer. CULLIN3KLHL22-RBX1 ligase mediated the polyubiquitination of GDH1 invitro. Together, these results enabled the establishment of a structural model for understanding the complete assembly of BTB-Kelch proteins with CULLIN3 and how together they recognize oligomeric substrates and target them for ubiquitination.

Hyperinsulinism/hyperammonemia syndrome caused by biallelic SLC25A36 mutation.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

On the human health benefits of microalgal phytohormones: An explorative in silico analysis

Phytohormones represent a group of secondary metabolites with different chemical structures, in which belong auxins, cytokinins, gibberellins, or brassinosteroids. In higher plants, they cover active roles in growth or defense function, while their potential benefits for human health protection were noted for some phytohormones and little explored for many others. In this study, we developed a target fishing strategy on fifty-three selected naturally occurring phytohormones covering different families towards proteins involved in key cellular functions related to human metabolism and health protection/disease. This in silico analysis strategy aims to screen the potential human health-driven bioactivity of more than fifty phytohormones through the analysis of their interactions with specific targets. From this analysis, twenty-eight human targets were recovered. Some targets e.g., the proteins mitochondrial glutamate dehydrogenase (GLUD1) or nerve growth factor (NGF) bound many phytohormones, highlighting their involvement in amino acid metabolism and/or in the maintenance or survival of neurons. Conversely, some phytohormones specifically interacted with some proteins, e.g., SPRY domain-containing SOCS box protein 2 (SPSB2) or Inosine-5′-monophosphate dehydrogenase 1 (IMPDH1), both involved in human immune response. They were then investigated with a molecular docking analysis approach. Our bioprospecting study indicated that many phytohormones may endow human health benefits, with potential functional role in multiple cellular processes including immune response and cell cycle progression.

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

Organic acids metabolism and GABA shunt involved in maintaining quality of Malus domestica by methyl jasmonate treatment

Apples (cv. Golden Delicious) were used as the materials to investigate methyl jasmonate (MeJA) dipping on quality parameters, organic acids metabolism and GABA shunt during storage at 21 ± 1 °C and 75 ± 5 % relative humidity. Results demonstrated that MeJA treatment reduced mass loss, respiratory intensity and ethylene release, and maintained higher flesh firmness and soluble solid content of apples. MeJA also decreased malic acid content, increased succinic and tartaric acids contents, and inhibited cytoplasmic aconitase (Cyt-ACO), NADP-malate (NADP-ME), phosphoenolpyruvate dehydrogenase (PEPC), mitochondrial citrate synthase (Mit-CS), glutamate dehydrogenase (GAD), and GABA transferase (GABA-T) activities in apples. NADP-isocitrate dehydrogenase (NADP-IDH), mitochondrial cis-aconitase (Mit-ACO), and cytoplasmic NAD-malate dehydrogenase (CytNAD-MDH) activities in apples were also enhanced by MeJA dipping. Moreover, MeJA dipping enhanced MdCytNAD-MDH and MdNADP-IDH expressions, and down-regulated MdGAD, MdGABA-T, MdNADP-ME, MdPEPC, MdCyt-ACO and MdMit-CS expressions in apples. These results suggest that MeJA dipping can maintain storage quality of “Golden Delicious” apples by regulating organic acids metabolism and GABA shunt.

Alterations in Inflammatory Cytokines and Redox Homeostasis in LPS-Induced Pancreatic Beta-Cell Toxicity and Mitochondrial Stress: Protection by Azadirachtin

Inflammation and redox imbalance are hallmarks of cancer, diabetes, and other degenerative disorders. Pathophysiological response to these disorders leads to oxidative stress and mitochondrial dysfunction by alterations and reprogramming in cellular signaling and metabolism. Pancreatic beta cells are very sensitive to the inflammatory and altered nutrient signals and hence play a crucial role in diabetes and cancer. In this study, we treated insulin-secreting pancreatic beta cells, Rin-5F, with the bacterial endotoxin, LPS (1 μg/ml) to induce an inflammatory response in vitro and then treated the cells with a known anti-inflammatory, anticancer and antioxidant phytochemical, azadirachtin (AZD, 25 µM for 24 h). Our results demonstrated lipid peroxidation and nitric oxide production causing increased nitro/oxidative stress and alterations in the activities of anti-oxidant enzymes, superoxide dismutase and catalase after LPS treatment. Pro-inflammatory responses caused by translocation of nuclear factor kappa B and release of inflammatory cytokines were also observed. These changes were accompanied by GSH-dependent redox imbalance and alterations in mitochondrial membrane potential and respiratory complexes enzyme activities leading to mitochondrial respiratory dysfunction, reduced ATP synthesis, and intrinsic caspase-9 mediated apoptosis. Caspase-9 was activated due to alterations in Bcl-2 and Bax proteins and release of cytochrome c into the cytosol. The activities of oxidative stress-sensitive mitochondrial matrix enzymes, aconitase, and glutamate dehydrogenase were also inhibited. Treatment with AZD showed beneficial effects on the recovery of antioxidant enzymes, inflammatory responses, and mitochondrial functions. GSH-dependent redox homeostasis also recovered after the treatment with AZD. This study may help in better understanding the etiology and pathogenesis of inflammation-induced disorders in pancreatic beta cells to better manage therapeutic strategies.

PNC2 (SLC25A36) Deficiency Associated With the Hyperinsulinism/Hyperammonemia Syndrome.

The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second-most common form of congenital hyperinsulinism, has been associated with dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP. To identify the underlying genetic etiology in 2 siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation. The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated. A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26-aa in-frame deletion in the first repeat domain of the protein, which abolishes transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content, which likely leads to a hyperactivation of glutamate dehydrogenase in our patients. We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.

Rapid metabolic and bioenergetic adaptations of astrocytes under hyperammonemia- a novel perspective on hepatic encephalopathy.

Hepatic encephalopathy (HE) is a well-studied, neurological syndrome caused by liver dysfunctions. Ammonia, the major toxin during HE pathogenesis, impairs many cellular processes within astrocytes. Yet, themolecular mechanisms causing HE are not fully understood. Here we will recapitulate possible underlying mechanisms with a clear focus on studies revealing a link between altered energy metabolism and HE in cellular models and in vivo. The role of the mitochondrial glutamate dehydrogenase and its role in metabolic rewiring of the TCA cycle will be discussed. We propose an updated model of ammonia-induced toxicity that may also be exploited for therapeutic strategies in the future.

Redirected nuclear glutamate dehydrogenase supplies Tet3 with \u03b1-ketoglutarate in neurons

Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.

In Situ Assessment of Hepatic Glutamate Dehydrogenase Activity Provides Novel Insights into Amino Acid Metabolism

The liver serves as a hub for amino acid metabolism, in particular concerning ammonia detoxification. The mitochondrial enzyme glutamate dehydrogenase (GDH) plays a central role this pathway, bridging amino acid deamination and glutamate handling toward urea formation. Numerous studies have investigated the function of GDH in the liver, pointing to complex allosteric regulations. However, typical measurements of GDH expression and activity in tissue extracts do not allow the assessment of the actual in situ enzymatic activity that relies on the preservation of intracellular organization. In this study, we developed an in situ redox-sensitive nitro blue tetrazolium (NBT) assay to investigate the hepatic GDH activity in cryopreserved liver sections. On the same section series, immunohistochemistry provided corresponding mapping of the expression of GDH and associated enzymes. The study was conducted in control floxed mice as well as in liver-specific GDH knockout mice (Hep-Glud1-/-). We evaluated the responses to different dietary protein intakes, i.e. mice fed a standard 20% protein diet versus 30% and 45% protein contents as high protein diets. Tissues of interest were collected from control floxed mice and Hep-Glud1-/- mice after 4 hours of fasting. Measured in liver lysates of control floxed mice, expression and activity of GDH were not significantly changed by the high protein diets. However, measured in situ, the NBT-based assay revealed that GDH activity was markedly increased by the high protein diets. In vivo, this was accompanied by an increase in plasma urea levels, while plasma ammonia levels remained unchanged. Surprisingly, Hep-Glud1-/- mice lacking liver GDH exhibited hyperammonemia, even under the standard 20% protein diet. In control floxed mice, immunohistochemistry on liver cryosections showed that GDH was evenly distributed within the tissue. However, the NBT assay performed on the same cryosections revealed hot spots of in situ GDH activity predominantly in hepatic perivenous area. These readouts were absent in Hep-Glud1-/- mice. Overall, our study shows that commonly used assessments of GDH function do not reflect in situ activity. Such activity is increased by a high protein diet, while expression levels are unchanged; measurements of GDH activity in liver extracts missing the in situ adaptation of the enzyme uncovered by the NBT assay.

Corrected and republished: comparative botanical and phytochemical studies of ambiguous medicinal plant species of wedelia and eclipta (fam. asteraceae) used in ASU systems of medicine with special reference to in-silico screening of hepatoprotective potential of marker wedelolactone with acetaminophen targets

Pharmacognosy Research,2021,13,1,54-67.DOI:10.4103/pr.pr_11_21Published:April 2021Type:Original ArticleAuthors:C. Arunachalam, R. Arunadevi, S. Murugamma, N. Monika, R. Susila, and K. N. Sunil Kumar Author(s) affiliations:C. Arunachalam, R. Arunadevi1, S. Murugammal2, N. Monika1, R. Susila3, K. N. Sunil Kumar4 Departments of Botany and 1Pharmacology, Captain Srinivasa Murthy Central Ayurveda Research Institute, (CCRAS, Ministry of AYUSH, Government of India, New Delhi), Anna Hospital Campus, Departments of 2Chemistry, 3Clinical Research and 4Pharmacognosy, Siddha Central Research Institute, (CCRS, Ministry of AYUSH, Government of India, Chennai), Anna Hospital Campus, Chennai, Tamil Nadu, INDIA Abstract:Post the publication, as there were multiple corrections that were missed earlier, the article is being corrected and republished. Comparative Botanical and Phytochemical Studies of Ambiguous Medicinal Plant Species of Wedelia and Eclipta (Fam. Asteraceae) Used in ASU Systems of Medicine with Special Reference to in-silico Screening of Hepatoprotective Potential of Marker Wedelolactone with Acetaminophen Targets. Context: In traditional medicine, Kesaraja (Ayurveda) or Manjal karisali (Siddha) is effective for jaundice. Aim: Three species of Asteraceae need to be studied for their therapeutic superiority of their intended claim. They are Wedelia chinensis (Osbeck) Merr. Philipp J., Wedelia trilobata (L.) Hitchc. and Eclipta prostrata (L.) L. (Asteraceae). The present study aimed to screen and characterize the potential species for therapeutic purpose. Materials and Methods: The whole plants, W. chinensis (Osbeck) Merr. W. trilobata (L.) Hitchc. and E. prostrata (L.) (Asteraceae) were collected and botanically identified. Preliminary phytochemical analysis and high-performance thin-layer chromatography finger printing with marker wedelolactone were done for the ethanolic extracts of these plants. Botanical and pharmacognostical diagnostic characters of the plants based on macro-morphological, micro-morphological and powder microscopical characterization were worked out. Comparative in-vitro antioxidant potential of ethanolic extracts of these plant species was carried out. Using ADMET SAR software, the pharmacokinetics of wedelolactone were predicted. Using Autodock 4.2 software, the binding energy of wedelolactone on targets of acetaminophen-induced hepatotoxicity namely PPAR-α, AMPK, JNK-1, EGFR, Nrf2, ALT, ALP, GGT, CAR, Frizzled receptor, FXR, ERK1, LXR, mitochondrial glutamate dehydrogenase, p53, mTOR C1, CYP1A2, CYP2E1, 5-lipoxygenase, thrombin, UCP1, GSK1, RXR and PXR was predicted. Results: All the three plant species were pharmacognostically and chemically different. W. chinensis was found to possess more antioxidant potential than the other two plants. The marker compound wedelolactone was not detected in W. trilobata. Wedelolactone passed the Lipinski's rule of five, and the docking analysis of wedelolactone confirmed high binding affinity toward PPAR-α, AMPK, Nrf2, CYP2E1, EGFR, JNK1, UCP-2, thrombin, 5-lipoxygenase, mTORC1, RXR, FXR, LXR, Frizzled receptor, GDH and Erk-1. Conclusion: Based on the above observations, we conclude that the presence of marker compound wedelolactone might have attributed the potency of W. chinensis and E. prostrata in counteracting acetaminophen toxicity when compared with W. trilobata. Keywords:Binding energy, botanical standardization, chromatographic fingerprinting, Docking, Hepatoprotective, in silico prediction, Lipinski's rule, Siddha, Therapeutic superiority, WedelolactoneView:PDF (6.36 MB)

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

ABSTRACTAstrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

Cancer proteome and metabolite changes linked to SHMT2.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

Changes in hypothermal stress-induced hepatic mitochondrial metabolic patterns between fresh water- and seawater-acclimated milkfish, Chanos chanos

Milkfish (Chanos chanos) is a tropical euryhaline species. It can acclimate to fresh water (FW) or seawater (SW) and be cultured in both. In winter, cold snaps cause huge losses in milkfish revenue. Compared to FW-acclimated individuals, SW-acclimated milkfish have better low-temperature tolerance. Under hypothermal stress, a stable energy supply is critical to maintain normal liver function. In this study, the levels of key mitochondrial enzymes (citrate synthase (CS) and cytochrome c oxidase (COX)) in milkfish livers were examined. The CS:COX activity ratio in FW milkfish significantly increased under hypothermal stress (18 °C) whereas ATP (the end product of aerobic metabolism) was downregulated. Therefore, the activities of the enzymes involved in mitochondrial amino acid biosynthesis (aspartate aminotransferase (AST) and glutamate dehydrogenase (GDH)) were evaluated to elucidate energy flow in milkfish livers under hypothermal stress. In FW milkfish, GDH activity was upregulated whereas AST activity was downregulated. Nevertheless, the levels of all the aforementioned enzymes did not significantly change in SW milkfish under hypothermal stress. In summary, we clarified the mechanism accounting for the fact that SW milkfish have superior low-temperature tolerance to FW milkfish and demonstrated that SW and FW milkfish have different and unique strategies for regulating energy flow.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Resolvin D1 attenuates liver ischaemia/reperfusion injury through modulating thioredoxin 2-mediated mitochondrial quality control.

Liver ischaemia and reperfusion (IR) injury is a sterile inflammatory response involving production of ROS. Mitochondrial homeostasis is maintained by mitochondrial quality control (QC). Thioredoxin (TRX) 2 is a key mitochondrial redox-sensitive protein. Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator, exerts anti-inflammatory and antioxidant activities. We investigated mechanisms of RvD1 protection against IR-induced oxidative damage to the liver, focusing on TRX2-mediated mitochondrial QC. Mice underwent partial warm IR. RvD1 was administered 1h before ischaemia and immediately prior to reperfusion. Human liver carcinoma HepG2 cells were exposed to hypoxia/reoxygenation and transfected with TRX2 siRNA. Immunohistochemistry, Western blotting and enzyme assays were used to follow changes in mitochondrial structure and function. RvD1 attenuated hepatocellular damage following IR, assessed by serum aminotransferase activities and histology. RvD1 reduced mitochondrial swelling, lipid peroxidation and glutamate dehydrogenase release. Impaired activities of mitochondrial complexes I and III were restored by RvD1. RvD1 enhanced expression of the mitophagy-related protein, Parkin and inhibited accumulation of PTEN-induced putative kinase 1. RvD1 restored levels of mitochondrial biogenesis proteins including PPARγ coactivator 1α, nuclear respiratory factor 1 and mitochondrial transcription factor A and mtDNA level. RvD1 attenuated the increase in levels of the mitochondrial fission-related protein, dynamin-related protein 1. IR reduced TRX2 levels while increasing TRX2 association with TRX-interacting protein. RvD1 attenuated these changes. The regulatory effects of RvD1 on mitochondrial QC were abolished by TRX2 knockdown. We suggest that RvD1 ameliorated IR-induced hepatocellular damage by regulating TRX2-mediated mitochondrial QC.

Glutamate dehydrogenase: role in regulating metabolism and insulin release in pancreatic β-cells.

Regulation of insulin release and glucose homeostasis by pancreatic β-cells is dependent on the metabolism of glucose by glucokinase (GK) and the influence of that activity on oxidative phosphorylation. Genetic alterations that result in hyperactivity of mitochondrial glutamate dehydrogenase (GDH-1) can cause hypoglycemia-hyperammonemia following high protein meals, but the role of GDH-1 remains poorly understood. GDH-1 activity is strongly inhibited by GTP, to near zero in the absence of ADP, and cooperatively activated ( n = 2.3) by ADP. The dissociation constant for ADP is near 200 µM in vivo, but leucine and its nonmetabolized analog 2-amino-2-norbornane-carboxylic acid (BCH) can activate GDH-1 by increasing the affinity for ADP. Under physiological conditions, as [ADP] increases GDH-1 activity remains very low until ~35 µM (threshold) and then increases rapidly. A model for GDH-1 and its regulation has been combined with a previously published model for glucose sensing that coupled GK activity and oxidative phosphorylation. The combined model (GK-GDH-core) shows that GK activity determines the energy state ([ATP]/[ADP][Pi]) in β-cells for glucose concentrations > 5 mM ([ADP] < 35 µM). As glucose falls < 5 mM the [ADP]-dependent increase in GDH-1 activity prevents [ADP] from rising above ~70 µM. Thus, GDH-1 dynamically buffers β-cell energy metabolism during hypoglycemia, maintaining the energy state and the basal rate of insulin release. GDH-1 hyperactivity suppresses the normal increase in [ADP] in hypoglycemia. This leads to hypoglycemia following a high protein meal by increasing the basal rate of insulin release (β-cells) and decreasing glucagon release (α-cells). NEW & NOTEWORTHY A model of β-cell metabolism and regulation of insulin release is presented. The model integrates regulation of oxidative phosphorylation, glucokinase (GK), and glutamate dehydrogenase (GDH-1). GDH-1 is near equilibrium under physiological conditions, but the activity is inhibited by GTP. In hypoglycemia, however, GK activity is low and [ADP], a potent activator of GDH-1, increases. Reducing equivalents from GDH dynamically buffers the intramitochondrial [NADH]/[NAD+], and thereby the energy state, preventing hypoglycemia-induced substrate deprivation.

Canagliflozin mediated dual inhibition of mitochondrial glutamate dehydrogenase and complex I: an off-target adverse effect

Recent FDA Drug Safety Communications report an increased risk for acute kidney injury in patients treated with the gliflozin class of sodium/glucose co-transport inhibitors indicated for treatment of type 2 diabetes mellitus. To identify a potential rationale for the latter, we used an in vitro human renal proximal tubule epithelial cell model system (RPTEC/TERT1), physiologically representing human renal proximal tubule function. A targeted metabolomics approach, contrasting gliflozins to inhibitors of central carbon metabolism and mitochondrial function, revealed a double mode of action for canagliflozin, but not for its analogs dapagliflozin and empagliflozin. Canagliflozin inhibited the glutamate dehydrogenase (GDH) and mitochondrial electron transport chain (ETC) complex I at clinically relevant concentrations. This dual inhibition specifically prevented replenishment of tricarboxylic acid cycle metabolites by glutamine (anaplerosis) and thus altered amino acid pools by increasing compensatory transamination reactions. Consequently, canagliflozin caused a characteristic intracellular accumulation of glutamine, glutamate and alanine in confluent, quiescent RPTEC/TERT1. Canagliflozin, but none of the classical ETC inhibitors, induced cytotoxicity at particularly low concentrations in proliferating RPTEC/TERT1, serving as model for proximal tubule regeneration in situ. This finding is testimony of the strong dependence of proliferating cells on glutamine anaplerosis via GDH. Our discovery of canagliflozin-mediated simultaneous inhibition of GDH and ETC complex I in renal cells at clinically relevant concentrations, and their particular susceptibility to necrotic cell death during proliferation, provides a mechanistic rationale for the adverse effects observed especially in patients with preexisting chronic kidney disease or previous kidney injury characterized by sustained regenerative tubular epithelial cell proliferation.

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers (anthelmintics). Benzimidazoles (BZ) are currently the only anthelmintic registered in the EU for use in controlling A. galli and there is an obvious risk of overuse of one drug class, selecting for resistance. Thus we developed a reference transcriptome of A. galli to investigate the response in gene expression before and after exposure to the BZ drug flubendazole (FLBZ). Transcriptional variations between treated and untreated A. galli showed that transcripts annotated as mitochondrial glutamate dehydrogenase and cytochrome P450 were significantly down-regulated in treated worms, whereas transcripts homologous to heat shock proteins (HSP), catalase, phosphofructokinase, and a multidrug resistance P-glycoprotein (PGP1) were significantly up-regulated in treated worms. Investigation of candidate transcripts responsible for anthelmintic resistance in livestock nematodes led to identification of several tubulins, including six new isoforms of beta-tubulin, and several ligand-gated ionotropic receptors and ABC-transporters. We discovered several transcripts associated with drug binding and processing genes, but further characterisation using a larger set of worms exposed to BZs in functional assays is required to determine how these are involved in drug binding and metabolism.